Project Summary Calcific aortic valve disease (CAVD) is common in the elderly. CAVD progression to significant aortic stenosis occurs over years in most patients. This indolent course affords the opportunity for therapeutic intervention to halt the disease progression at an early stage. However, limited knowledge of mechanisms underlying CAVD progression impedes the development of effective pharmacological therapies. In the current funding period, our extensive studies have identified signaling mechanisms mediated by innate immune receptors, particularly TLR2 and TLR4, as regulators of the fibrogenic and osteogenic activities in human aortic valve interstitial cells (AVICs). We also discovered that oxidized low-density lipoprotein (oxLDL) and extracellular matrix (ECM) protein matrilin 2 function as damage-associated molecular patterns (DAMPs) in human AVICs, prompting differential cell activation with elevated fibrogenic and osteogenic activities. In addition we observed that human monocytes activated by TLR2 up-regulate AVIC expression of pro-fibrogenic and pro-osteogenic mediators via a paracrine mechanism. Planned studies in this renewal application will test the central hypothesis that distinct molecular mechanisms triggered by AVIC innate immune response elevate cellular and valvular fibrogenic and osteogenic activities through induction of cell activation and/or myofibroblastic transition. We further hypothesize that down- regulation of AVIC innate immune response has therapeutic potential to suppress valvular fibrosis and calcification. These hypotheses are based on our recent observation that oxLDL elevates AVIC fibrogenic and osteogenic activities through a myofibroblast transition-independent mechanism, whereas soluble matrilin 2 elevates these activities in human AVICs through inducing myofibroblastic transition. Moreover, we discovered that human AVICs express anti-aging protein Klotho. This anti-aging protein is capable of modulating AVIC innate immune response. Interestingly, Klotho levels are much lower in aortic valve tissues and AVICs from patients with CAVD, and Klotho insufficiency exacerbates valvular fibrogenic and osteogenic activities. This grant application will address: (1) the mechanisms underlying AVIC fibrogenic and osteogenic activities, (2) the mechanism by which Klotho insufficiency exacerbates AVIC fibrogenic and osteogenic activities, (3) anti- inflammatory approaches to up-regulate Klotho expression in aortic valve and (4) the therapeutic potential of Klotho to alleviate valvular fibrosis and calcification. This proposed work will provide mechanistic insights into the pathobiology of CAVD progression and identify therapeutic targets for pharmacological intervention to halt CAVD progression.